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Primer heterodimer muammosi

Primer heterodimer muammosi



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Men primer3PLUS -da primerlarimni loyihalashtiraman va ularni Oligoanalyzer3.1 -da tahlil qilaman, menda katta muammo shundaki, men NCBI -Primer BLAST -da o'ziga xosligi uchun juda yaxshi natijaga erishdim, lekin o'zim yoki hetrodimer uchun juda salbiy DG -7 yoki undan past -13. DMSO yoki Formamid qo'shish orqali ularni PCR uchun ishlatish mumkinmi? boshqa qanday usul foydali bo'lishi mumkin?


Asosiy qo'ng'iroq, o'qish xaritasi va qamrov tahlili

Foydalanish mumkin bo'lgan ketma-ketlik

Poliklonal Internet-provayderlarga qo'shimcha ravishda, Torrent ham past sifatli va primer dimer o'qishlarini filtrlaydi va "foydalanish mumkin bo'lgan ketma-ketlik" poliklonal, past sifatli va primer dimer filtrlaridan o'tgan Kutubxona provayderlarining ulushi. Past sifatli filtr noaniq qo'ng'iroqlar bilan o'qishni yo'q qiladi, hech bo'lmaganda ularning ba'zilari shablon ketma -ketligining maqbul darajasidan past bo'lgan provayderlar tomonidan ishlab chiqariladi. Dastur shuningdek, 3' adapter ketma-ketligi uchun o'qishlarni skanerlaydi. Uzunligi 8 bp dan kam bo'lgan kutubxona parchalari primer dimerlari hisoblanadi va filtrlanadi.


Yadro retseptorlari

Neil J. Mckenna, David D. Mur, Endokrinologiyada (Oltinchi nashr), 2010 yil

SHP va DAX-1

Kichik heterodimer sherigi (SHP) va dozaga sezgir jinsiy reversal-adrenal gipoplaziya X xromosomasida tug'ma kritik mintaqa (DAX-1) yadro retseptorlari DNK-bog'lash domeniga ega bo'lmagan noyob etim retseptorlari. SHP boshqa bir qator yadro retseptorlari bilan bevosita ta'sir o'tkazishi va transkripsiyani faollashtirish qobiliyatini inhibe qilishi mumkin. 45 Yuqorida ta'kidlab o'tilganidek, SHP nokautlari bilan natijalar safro kislotasi biosintezining salbiy qayta regulyatsiyasi uchun FXR-ga bog'liq yo'lda SHP uchun taklif qilingan o'ziga xos rolni qo'llab-quvvatladi. Qizig'i shundaki, bu jarayonning qo'shimcha ortiqcha mexanizmlari mavjud, chunki SHP nol sichqonlari sintetik FXR agonistiga javoban kutilgan repressiya yo'qolishini ko'rsatadi, lekin asosan yuqori darajadagi o't kislotalarining repressiv ta'sirini saqlaydi. 46,47

Faqat ligand-bog'lovchi domendan iborat bo'lgan SHPdan farqli o'laroq, DAX-1 turli xil DNK-bog'lanish faoliyati bilan bog'liq bo'lgan qo'shimcha N-terminal domenini o'z ichiga oladi, ammo bu potentsial funktsiyaning ahamiyati noaniqligicha qolmoqda. 48 Odamning funktsiyalarini yo'qotishi DAX1 gen gipogonadotropik gipogonadizm bilan bog'liq bo'lgan konjenita adrenal gipoplaziyaning X-bog'langan shaklini keltirib chiqaradi. SHP singari, DAX-1 transkripsiyali repressor vazifasini bajaradi va bu repressiya funktsiyasining yo'qolishi bu fenotipga to'g'ri keladi deb ishoniladi. DAX-1 ning transkripsiyaviy maqsadlari noma'lum bo'lib qolmoqda, biroq bir nechta dalillar qatori, jumladan, to'g'ridan-to'g'ri o'zaro ta'sir va shunga o'xshash ifoda shakllari, u SF-1 funktsiyasini modulyatsiya qilishini ko'rsatadi. 49


Primer heterodimer muammosi - Biologiya

DNKning eng oddiy komponenti nukleotiddir. Aniqroq aytganda, bu deoksiribonukleotiddir. U shakarning 5' uglerodiga biriktirilgan fosfat guruhidan tashkil topgan orqa miyaga ega. Bular DNKning umurtqa pog'onasini yoki zinapoyaning yon tomonlarini hosil qiladi. DNK kodi azotli asoslar bilan belgilanadi. Va bular adenin, timin, guanin yoki sitozdan iborat bo'lishi mumkin

Har bir nukleotid boshqasi bilan fosfodiester aloqasi deb ataladigan narsa bilan bog'langan. Va DNKning yo'nalishi bor. DNK 5-boshdan 3-bosh yoʻnalishgacha oʻqiladi. Buning ma'nosi shundaki, DNK biz o'qigan kitobga o'xshab o'qiladi. U ipning 5 -boshidan boshlanadi. Bu fosfat guruhidan boshlanadigan ip. Va keyin DNK 3 asosiy yo'nalishga yoki DNKning shakar uchiga qarab o'qiladi.

1950 -yillarga qadar DNK haqida ko'p narsa ma'lum emas edi. Biz DNK hayot kodi ekanligini bilardik. Ammo biz bu qanday ishlashini bilmas edik. Biz faqat shakar, fosfat va to'rtta azotli asosdan yasalganini bilardik. DNK bo'yicha asosiy ishlarning ko'pini Jeyms Uotson va Frensis Krik bajargan. 1953 yilda Uotson va Krik DNK tuzilishini aniqladilar. Hamkorligi davomida Uotson va Krik uchta xususiyatni taklif qilishdi.

DNK molekulasining ikkita umurtqa suyagi yonma-yon joylashganligi ma'lum edi. Biroq, ular nazariy jihatdan ikki xil yo'lni birlashtirishi mumkin edi. Ular bir xil yo'nalishda turishi mumkin edi, ya'ni zinapoyaning har ikki tomoni bir xil yo'nalishda 5 boshdan 3 boshgacha bo'ladi. Ammo Uotson va Krik DNKning ikkita zanjiri antiparallel ekanligini taklif qilishdi. Bu ularning perpendikulyar ekanligini anglatmaydi. Aksincha, bu chiziq yonma-yon turishini anglatadi, lekin DNK magistralining yo'nalishi ikki xil yo'nalishda ketadi. Va agar bu to'g'ri bo'lsa, Uotson va Krik azotli asoslarning juftligi bir-birini to'ldiruvchi ekanligini taklif qilishdi. Ya'ni, adenin timin bilan, guanin esa sitozin bilan bog'lanadi. Agar DNK antiparalel emas, balki parallel bo'lsa, nazariy jihatdan, azotli asoslar bir -biriga bog'langan bo'lishi mumkin. Bu A bilan A, C bilan C va hokazo.

Uotson va Krik keyinchalik Nobel mukofotiga sazovor bo'lishdi. Ularning kashfiyoti ko'p jihatdan ayol kimyogar Rozalind Franklinning ishiga bog'liq edi, uning tadqiqotlari uning bilimi yoki ruxsatisiz ishlatilgan. Eng yomoni, u o'z ishi uchun maqtovga sazovor bo'lmadi. U DNK tuzilishini aniqlaydigan muhim nashrga mualliflik qilmagan. Aynan Franklinning DNK molekulasi surati Uotson va Krik boshchiligidagi ilmiy inqilobni keltirib chiqardi. Uotson bu suratni birinchi marta ko'rgani haqida shunday dedi: "Mening jag'im ochilib, yurak urishim tezlasha boshladi". Fotosuratda birinchi marta DNKning muhim tuzilishi-juft spiral shakli ko'rsatildi, bu uning replikatsiya usulini ham ko'rsatdi. Franklinning fotografik mahorati kashfiyotni amalga oshirishga imkon berdi. U boshqa odamlar jurnalda chop etilgan maqolaga asoslanib, uning tadqiqotidan foydalanayotganini bilmas edi Tabiat. U Nobel mukofotiga ham qo'shilmasdi. Biroq, bu Franklinni e'tibordan chetda qoldirgani uchun emas, balki u o'lganligi uchun. Mukofot o'limdan keyin berilmaydi. Franklinga 1956 yilda 37 yoshida tuxumdon saratoni tashxisi qo'yilgan va u ikki yildan so'ng vafot etgan (ehtimol, u laboratoriya ishlarida radiatsiya ta'sirida bo'lgani uchun).

Vaziyat Rozalind Franklin bilan qanchalik shiddatli bo'lsa, Jeyms Uotson va Frensis Krik haqiqatan ham inqilobiy olimlar edi. Uotson va Krik DNKning ikkita ipi yangi DNK iplarini ishlab chiqarish uchun shablon (yoki naqsh) bo'lib xizmat qilishini taklif qilishdi. Shunday qilib, DNKni qayta -qayta nusxalash mumkin edi. Hayot shuni talab qiladi. Ularning ta'kidlashicha, bu iplar ushbu qo'shimcha tayanch juftligiga ko'ra ko'chiriladi. Boshqacha qilib aytganda, DNK molekulasi ochiladi va azotli asos juftlari ularning komplementi bilan mos keladi (Ts bilan Cs va Gs bilan).

DNK replikatsiyasi gipotezalari

DNKning tuzilishi o'rnatilgandan so'ng, keyingi asosiy ish uning qanday takrorlanishini tushunish edi. Va DNKni ko'paytirishning uchta mumkin bo'lgan usuli bor. Bular DNK replikatsiyasi uchun muqobil gipotezalar sifatida tanilgan. Yarim konservativ replikatsiya gipotezasi, ota-ona DNKining ajralib chiqishini va har bir ipning shablon bo'lib xizmat qilishini taxmin qiladi, unda har bir eski ipdan yangi nusxa olinadi. Shunday qilib, rasmda pushti iplar ota -onaning DNK zanjirlarini, sariq ip esa nusxalangan xromosomalarni (qiz xromosomalarini) ifodalaydi. Pushti iplar ajralib chiqadi, nusxasi tayyorlanadi va keyin har bir yangi qo'sh DNK zanjirida ota-ona va qiz zanjiri mavjud.

Konservativ replikatsiyada ota -ona DNKi yangi molekula sintezi uchun shablon sifatida ishlatiladi. Boshqacha qilib aytadigan bo'lsak, ikkala ota-ona DNK zanjiri nusxalanadi va biz ikkita DNK zanjiriga ega bo'lamiz, biri asl ota-ona DNKsi, ikkinchisi esa DNKning ota-ona qo'sh zanjirining to'liq nusxasi.

DNK replikatsiyasining boshqa imkoniyati dispersiv replikatsiya deb nomlanadi. Ushbu modelda yangi DNK molekulalari ota -ona va qiz DNK segmentlarining birikmasidir. … Shunday qilib, DNK qanday replikatsiya qilinadi. Biz aqlli tajriba natijalari asosida bilamiz.

1958 yilda Metyu Meselson va Franklin Stal bu gipotezalarning qaysi biri qo'llab -quvvatlanishini aniqlash uchun tajriba ishlab chiqdilar. Azot DNKning asosiy tarkibiy qismidir. 14N azotning eng keng tarqalgan izotopidir, ammo og'irroq (ammo radioaktiv bo'lmagan) 15N izotopi bo'lgan DNK ham ishlaydi. Meselson va Stahl E. coli ni «og'ir» azotda (15N) o'stirishdi. Bu DNKda atigi 15N bo'lgan E. coli zanjirini yaratish edi. 15N yilda ko'p avlodlar davomida E. coli o'sgandan so'ng, og'ir azotli E. coli oddiy azotli muhitda 14N o'stirildi. Keyin, ular bir necha avlodlar davomida E. coli ni kuzatib borishdi va har bir avlodda 15N va 14N nisbatlarini aniqlay olishdi. Ushbu nisbatni solishtirish tadqiqotchilarga ushbu muqobil gipotezalarning qaysi biri qo'llab-quvvatlanishini aniqlashga imkon beradi. Keling, bu qanday sodir bo'lishini ko'rib chiqaylik.

Yangi avlod E. coli voyaga etganida, DNKning yarmi ota -ona, DNKning yarmi esa nusxa bo'lardi. Meselson va Vahl 15N va 14N miqdorini o'lchab, ota-ona va qiz xromosomalarining foizini aniqlashlari mumkin edi. Bu DNK replikatsiyasining barcha muqobil gipotezalariga to'g'ri keladi. Biroq, 2 avloddan keyin bu nisbatlar boshqacha bo'lardi. Bu uchta modelning har biri "15N" DNKning replikatsiyadan keyin hosil bo'lgan molekulalarda tarqalishi haqida turlicha bashorat qiladi. Konservativ gipotezada, replikatsiyadan so'ng, bitta molekula butunlay saqlanib qolgan "15N" molekulasi, ikkinchisi esa yangi sintez qilingan DNKdir. Yarim konservativ gipoteza shuni ko'rsatadiki, replikatsiyadan keyin har bir molekulada bitta eski va bitta yangi zanjir mavjud. Bu shuni anglatadiki, ikkinchi avlodning ½ qismi past zichlikdagi DNK (to'liq 14N dan tashkil topgan), yarmida esa DNKning oraliq zichligi bo'ladi. Oraliq zichlik ½ 14N DNK va ½ 15N DNKga ega bo'ladi.

Konservativ gipoteza shuni ko'rsatadiki, DNKning har bir juft ipi har bir avlodning o'ziga xos nusxasini yaratadi. Agar DNK shu tarzda ko'chirilsa, 15N va 14N nisbati boshqacha bo'lar edi. Ikkinchi avlodda DNK ¼ yuqori zichlikka ega bo'lardi (ya'ni DNK 100% 15N DNKdan), namunaning ¾ past zichlikli DNK (ya'ni 14N DNK). Tarqatilgan model har bir yangi molekulaning har bir ipi eski va yangi DNK aralashmasidan iborat bo'lishini taxmin qiladi. Agar DNK dispersiv replikatsiya orqali nusxa ko'chirilsa, har bir avlod o'rta zichlikdagi DNKdan iborat bo'ladi. Ya'ni, barcha DNK 15N DNK va 14N DNK qismlaridan iborat bo'ladi.

E. coli 15N bo'lgan muhitda bir necha avlod uchun o'stirildi. Bu hujayralardan DNK olinib, tuz zichligi gradiyenti bo'yicha santrifüjlanganda, DNK zichligi tuz eritmasiga teng bo'lgan nuqtadan ajralib chiqadi. 15N muhitda o'sgan hujayralarning DNK zichligi oddiy 14N muhitda o'sgan hujayralarga qaraganda yuqori edi. Undan keyin, E. coli DNKida faqat 15N bo'lgan hujayralar 14N muhitga o'tkazildi va bo'linishga ruxsat berildi. DNK vaqti -vaqti bilan chiqariladi va 14N DNK va 15N DNK bilan taqqoslanadi. Bitta replikatsiyadan so'ng, DNK oraliq zichlikka yaqin ekanligi aniqlandi. Konservativ replikatsiya natijasida yuqori va quyi zichlikdagi DNKlar teng bo'ladi (lekin DNK oraliq zichligi yo'q), konservativ replikatsiya chiqarib tashlandi.

Biroq, bu natija yarim konservativ va dispersiv replikatsiyaga mos keldi. Yarimkonservativ replikatsiya natijasida bitta 15N DNK va 14N DNK dan iborat boʻlgan ikki zanjirli DNK hosil boʻladi, dispersiv replikatsiya esa har ikkala zanjirda 15N va 14N DNK aralashmasi boʻlgan ikki zanjirli DNK hosil boʻladi, ularning har biri DNK shaklida paydo boʻladi. O'rta zichlikdagi DNK. Mualliflar replikatsiya davom etar ekan, hujayralarni namuna olishni davom ettirdilar. Ikki replikatsiya tugagandan so'ng hujayralardan olingan DNK ikki xil zichlikdagi teng miqdordagi DNKdan tashkil topgan bo'lib, ulardan biri 14N muhitda faqat bitta bo'linish uchun o'sgan hujayralar DNKining oraliq zichligiga to'g'ri keladi, ikkinchisi o'sgan hujayralar DNKiga to'g'ri keladi. faqat 14N muhitda. Bu dispersiv replikatsiyaga mos kelmas edi, bu esa bir avlod hujayralarining oraliq zichligidan past bo'lgan yagona zichlikka olib kelishi mumkin edi, lekin baribir faqat 14N DNK muhitida o'stirilgan hujayralardan yuqori, chunki asl 15N DNK teng ravishda bo'lingan bo'lar edi. barcha DNK zanjirlari orasida. Natija yarim konservativ replikatsiya gipotezasiga mos keldi.

Meselson va Stahl har bir ota-onaning DNK zanjiri to'liq nusxalanganligini ko'rsatdi, bu DNKning yarim konservativ gipoteza orqali ko'payishini anglatadi. Biroq, ular mexanizmni bermadilar. DNK polimerazasining kashf etilishi DNK sintezi mexanizmiga yo'l ochdi. DNK polimeraza fermentining kashf qilinishi DNKning replikatsiya mexanizmini aniqroq tushunishga olib keldi. Nukleotidlar faqat yadro magistralining 3' uchiga qo'shilishi aniqlandi. Shunday qilib, DNK har doim 5 dan 3 gacha yo'nalishda sintezlanadi. Boshqacha qilib aytganda, DNK kitob kabi old tomondan orqaga o'qiladi (hech qachon orqadan oldinga).

Replikatsiyani boshlash

DNK xromosomalardagi replikatsiya pufakchalarida takrorlanadi. Va sintez ikki yo'nalishda davom etadi. Replikatsiya 5 dan 3 gacha yo'nalishda sodir bo'lganligi uchun va DNK antiparallel bo'lgani uchun sintez har ikki yo'nalishda ham sodir bo'ladi. Bakteriyalar eukaryotlarda replikatsiyaning yagona kelib chiqishiga ega, DNK replikatsiyaning bir necha kelib chiqishida takrorlanadi. Keling, DNK qanday replikatsiya qilinishini batafsil ko'rib chiqaylik. DNK replikatsiyani ochish, ochish va replikatsiya uchun tayyorlash orqali boshlaydi. DNK juft spirali spiral deb nomlanuvchi ferment tomonidan ochiladi. Bu DNKning ikkita ipi orasidagi bog'lanishni buzadigan maxsus ferment. Keyin DNK spiral stabillashadi. Bu shuni anglatadiki, bitta zanjirli DNKni bog'lovchi oqsillar deb ataladigan maxsus oqsillar avtomatik ravishda qayta bog'lanishiga yo'l qo'ymaslik uchun ochilgan DNK zanjirlariga biriktiriladi. Tasavvur qiling -a, sizning qo'llaringizdan boshqa hech narsa bo'lmagan holda, metall ipga o'ralgan. Siz tezda sezasizki, bo'shashgan odamning bo'shashishi jiddiy zo'riqishni keltirib chiqaradi. Siz bo'shashish jarayonini osonlashtirish uchun ba'zi vositalardan foydalanishingiz mumkin.

DNK bu muammo bilan kurashish uchun maxsus oqsildan foydalanadi. Bu oqsil topoizomeraza deb ataladi va u replikatsiya vilkasidan pastda DNKni kesadi va qayta qo'shiladi, natijada spiralning ochilishi natijasida paydo bo'ladigan taranglikni ketkazadi. Boshlanishning oxirgi bosqichida DNK polimeraza primerlanadi. Primaza deb nomlanuvchi maxsus oqsil birinchi fosfodiester aloqasini hosil qilish uchun nukleotid bo'lishi mumkin bo'lgan 3' gidroksil guruhini ta'minlash orqali replikatsiya jarayonini boshlaydi. Endi DNK replikatsiya qilishga tayyor.

Replisoma DNK replikatsiyasini amalga oshiradigan murakkab molekulyar mashinadir. U ikkita DNK polimeraza kompleksidan iborat bo'lib, ulardan biri etakchi zanjirni, ikkinchisi esa orqada qolgan zanjirni sintez qiladi.

Har bir bo'linuvchi nukleoid ikki tomonlama replikatsiya uchun ikkita replizomani talab qiladi. Ikkala nashr ham hujayraning o'rtasida ikkala vilkada ham replikatsiyani davom ettiradi. Nihoyat, tugatish joyi takrorlanganda, ikkita replikom DNKdan ajralib turadi.

Etakchi shablon - bu doimiy ravishda takrorlanadigan DNK ipi. Bu replikatsiya vilkasi tomon doimiy ravishda polimerizatsiya qilinayotgan ipdir. Barcha DNK sintezi 5'-3 'da sodir bo'ladi. Qolgan ip o'sayotgan vilkaning harakatiga qarama -qarshi yo'nalishda o'sadi. U replikatsiya vilkasidan uzoqda o'sadi va u uzluksiz sintezlanadi. U ipdan uzoqda sintezlangani va vilkaning orqasida qolganligi uchun uni ortda qolgan ip deb atashadi.

Ortiqcha ipning sintezi

Ip replikatsiya vilkasidan uzoqlashib borayotganligi sababli, uni bo'laklarga bo'lish kerak, chunki primaza (RNK primerini qo'shadi) primer qo'shish uchun vilka ochilguncha kutishi kerak. Primaza biriktirilgach, DNK polimeraza primerning 3' uchiga asoslar qo'shishni boshlaydi va replikatsiya vilkasidan uzoqroqda nukleotidlarni qo'shib, DNK segmentlarini hosil qiladi.

Kechiktirilgan ipda hosil bo'lgan DNKning bu bo'laklari Okazaki bo'laklari deb ataladi. Asl DNKning orqada qolgan ipda yo'nalishi doimiy sintezni oldini oladi. Natijada, ortda qolgan ipning replikatsiyasi etakchi ipni takrorlashdan ko'ra murakkabroqdir. Bu jarayon DNKning keyingi ochilishi bilan vilkada takrorlanadi. Qolgan narsa - DNK shablonidagi Okazaki bo'laklari. Nihoyat, DNK segmentlari bir -biriga DNK ligazasi bilan biriktirilgan.


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Muammo: RNK primeri DNKda sintezlanishi uchun quyidagi fermentlardan qaysi biri zarur:a. Gyraseb. DNK polimeraza Ic. Ligazlangan DNK. Primase

DNKda RNK primerini sintez qilish uchun quyidagi fermentlardan qaysi biri zarur:

tez-tez so'raladigan savollar

Ushbu muammoni hal qilish uchun qanday ilmiy tushunchani bilishingiz kerak?

Bizning o'qituvchilarimiz ushbu muammoni hal qilish uchun DNK replikatsiyasiga kirish tushunchasini qo'llash kerakligini ta'kidladilar. Siz DNK replikatsiyasiga kirishni o'rganish uchun video darslarni ko'rishingiz mumkin. Yoki sizga DNK replikatsiyasi amaliyotiga ko'proq kirish kerak bo'lsa, siz DNK replikatsiyasiga kirish amaliyoti muammolarini ham mashq qilishingiz mumkin.

Bu muammoning murakkabligi nimada?

Bizning o'qituvchilarimiz qiyinchiliklarni baholadilarDNKda RNK primerini sintez qilish uchun. past qiyinchilik kabi.

Bu muammoni hal qilish uchun qancha vaqt ketadi?

Biologiya bo'yicha mutaxassisimiz Keytlin bu muammoni hal qilish uchun 3 daqiqa 30 soniya vaqt sarfladi. Siz yuqoridagi video tushuntirishda ularning qadamlarini bajarishingiz mumkin.

Bu muammo qaysi professorga tegishli?

Bizning ma'lumotlarga asoslanib, biz bu muammo UCF professori Flores va#x27 sinflari uchun dolzarb deb o'ylaymiz.


KIRISH

P54nrb (sichqonchada NONO) yadro ichidagi ko'plab jarayonlarda, shu jumladan transkripsiyani tartibga solish, biriktirish, DNKni ajratish, gipereditlangan ikki tarmoqli RNKni yadroda ushlab turish, virusli RNKni qayta ishlash, hujayra proliferatsiyasini nazorat qilish va sirkadiyalik ritmni saqlash (Shav-Tal) bilan bog'liq. va Zipori, 2002 Braun va boshqalar., 2005). P54nrb ko'p va hamma joyda mavjud, bir va ikki zanjirli RNK va DNKni bog'lashi mumkin va o'ziga xos karbonat angidraz faolligiga ega. Dastlab, p54nrb PSF (Zhang va boshqalar.P54nrb ga o'xshash ketma -ketlik gomologiyasiga ega bo'lgan oqsil va ko'plab hisobotlarda p54nrb va PSF p54nrb topilgan ko'plab yadro komplekslarida kopurifikatsiya qilinganligi ko'rsatilgan.

P54nrb yadro ichida barcha xilma -xil harakatlarini qanday amalga oshirishi asosiy jumboqdir. Bu erda p54nrb ning fosforillanish holati va yadro ichidagi joylashuvi juda muhim deb taxmin qilingan (Shav-Tal va Zipori, 2002). Biz ilgari p54nrb paraspeckles, yangi yadro osti bo'linmasi boyitilgani haqida xabar bergan edik (Fox va boshqalar., 2002). Paraspeckles dastlab HeLa hujayralarida aniqlangan, lekin ular birlamchi hujayralarda, boshqa transformatsiyalangan hujayralar liniyalarida va to'qima bo'limlarida ham uchraydi (Fox va boshqalar., 2002). Sutemizuvchilar xujayralari odatda interkromatin bo'shlig'ida 2-20 parantezlarni o'z ichiga oladi, odatda splicing dog'lar yaqinida. Paraspeckle Protein 1 (PSP1) - bu p54nrb va PSF (Fox) bilan ketma -ket o'xshashlikni taqsimlaydigan parashekllar uchun marker. va boshqalar., 2002). PSP1 odatda paragraflarda to'planadi va nukleoplazmada tarqalgan. Biroq, RNK Polimeraza (Pol) II transkripsiyasi dori bilan davolash orqali inhibe qilinganida, PSP1 parantezlarni qoldiradi va yadroda to'planib, "perinukleolyar qopqoqlar" deb ataladigan yarim oy shaklidagi tuzilmalarda to'planadi. FLIP (foto oqartirishdan keyin lyuminestsent yo'qotish) shuni ko'rsatadiki, PSP1 molekulalari transkripsiya faol bo'lganda paraspeckles va nucleoli o'rtasida doimiy ravishda aylanib yurishadi (Fox va boshqalar., 2002). Xuddi shunday, PSP1 va p54nrb paraspeckle oqsillari ham odam yadrosi proteomida uchraydi (Andersen va boshqalar., 2002) va p54nrb transkripsiyani inhibe qilganda bir xil perinukleolyar qopqoqlarda to'planadi. Paraspecklesda bo'lishidan tashqari, PSP1 funktsiyasi haqida kam narsa ma'lum. Sichqoncha va odamda kamida ikki xil izoform - PSP1a (HeLa hujayralaridagi asosiy izoform, "PSP1" deb nomlangan) va PSP1b aniqlangan. va boshqalar., 2002 yil Myojin va boshqalar., 2004) va to'qimalarga xos bo'lishi mumkin (Myojin va boshqalar., 2004).

P54nrb, PSP1 va PSF sequence50% ketma-ketlik identifikatoriga ega, biroq PSF uni PSP1 va p54nrbdan ajratib turadigan qo'shimcha katta N-terminalli domenga ega. Asosiy o'xshashlik "DBHS" deb ataladigan narsada joylashgan (Drozofilabehaver va human splicing) domeni, ikkita RRM motifini o'z ichiga oladi, so'ngra zaryadlangan oqsil-oqsil o'zaro ta'sir moduli. Uy xo'jaligi roliga mos ravishda, uchta oqsil ham hamma joyda ifodalanadi va umurtqali hayvonlarda saqlanadi. Kabi umurtqasiz hayvonlar turlari Drosophila melanogaster, Caenorhabditis elegans, va chivinda faqat p54nrb/PSF/PSP1 oilasini ifodalovchi bitta gen bor. In D. melanogaster, NONA, ko'zning rivojlanishi va xulq -atvori bilan shug'ullanadi (Jons va Rubin, 1990) va p54nrb (jigarrang) kabi sirkadiyalik ritmni saqlashda rol o'ynaydi. va boshqalar., 2005). In Chironomus tentanlar, homolog Hrp65 bo'lib, u uchta izoformada ifodalanadi (Miralles va Visa, 2001) va ulardan biri, Hrp65-2, aktinni bog'laydi (Miralles) va boshqalar., 2000 Percipalle va boshqalar., 2003). Hrp65 Balbiani halqasining transkriptlari va DBHS domenining C-terminal qismi orqali o'zini o'zi bog'laydigan tolalarni o'z ichiga oladi. va boshqalar., 2003).

PSP1 va p54nrbdan tashqari, RNK bilan bog'laydigan yana ikkita oqsil, HeLa hujayralarida parasekllarga joylashadi. Birinchidan, PSP2 (SIP/CoAA), shuningdek, ikkita RRM motivini o'z ichiga oladi va steroid retseptorlariga bog'liq transkripsiyani tartibga soluvchi (Ivasaki) transkripsiyaviy corepression va koaktivatsiya komplekslarida topilgan. va boshqalar., 2001 yil Auboeuf va boshqalar., 2004). PSP1/PSF/p54nrb oilasi va PSP2 o'rtasidagi yana bir bog'liqlik ularning Androgen retseptorlari bilan bog'lovchi oqsillarni (Ishitani) izlashda o'zaro tozalanishidir. va boshqalar., 2003). Nihoyat, mRNK 3′-end jarayonining birinchi bosqichida ishtirok etgan omil, CFI(m)68, qisman parantezlar bilan birgalikda joylashgan o'choqlarda to'planishi haqida xabar berilgan (Dettwiler). va boshqalar., 2004).

P54nrb / PSP1 / PSF oqsillari oilasining paraspeckle lokalizatsiyasini tavsiflash uchun biz hujayra tsikli davomida paraspeckle shakllanishi va saqlanishini tahlil qildik. Biz PSP1 p54nrb bilan yangi heterodimer hosil qilishini va RNKga bog'liq holda parantezlarga qaratilganligini aniqlaymiz, uning perinukleolyar qopqoqlarda to'planishi esa RNKni talab qilmaydi.


7.1: Polimeraza zanjiri reaktsiyasiga umumiy nuqtai

  • Clare M. O & rsquoConnor hissasi
  • Boston kollejining faxriy professori (biologiya).

Polimeraza zanjirli reaktsiyasi (PCR) molekulyar biologiyada inqilob qildi. PCR yordamida tadqiqotchilar DNK sekanslarini juda oz miqdorda kuchaytirish vositasiga ega bo'lishdi
DNK shablonini. Darhaqiqat, odatdagi PCR reaktsiyasida bitta DNK molekulasidan milliardlab nusxalarni sintez qilish mumkin. PCRning rivojlanishi DNK -polimerazalar bo'yicha olib borilgan tadqiqotlar va oqsillarning ko'p qismini denatatsiya qiladigan issiqlik bilan ishlov berishga bardoshli termostabil DNK -polimerazalarning kashf qilinishi natijasida paydo bo'ldi. va boshqalar., 1988). Bugungi kunda PCR DNK molekulalarini tahlil qilish va yangi rekombinant molekulalarni yaratish uchun keng qo'llaniladigan standart texnikadir.

Termostabil DNK polimerazalari PCR uchun markaziy hisoblanadi. Birinchi ishlatiladigan PCR tavsifi
dan DNK polimeraza E. coli, denaturatsiyaga uchragan va har turdan keyin o'zgartirilishi kerak edi
DNK sintezi (Sakay va boshqalar., 1985). ni almashtirish orqali protsedura ancha yaxshilandi E.
coli
dan DNK polimeraza bilan polimeraza Thermus aquaticus, o'sadigan bakteriya
Yellouston milliy bog'idagi termal buloqlarda. The T. aquaticus DNK polimeraza yoki Taqpolimeraza, 70-75 ̊C haroratda eng yaxshi ishlaydi va 90 va 778C dan yuqori haroratlarda uzoq muddatli inkubatsiyaga bardosh bera oladi. Bir necha yil ichida,Taq polimeraza klonlangan va ortiqcha ifodalangan E. coli, uning mavjudligini sezilarli darajada kengaytiradi. Bugungi kunda PCR uchun mavjud bo'lgan polimerazalarni tanlash keskin oshdi, chunki boshqa termofil organizmlarda yangi DNK polimerazalar aniqlandi va genetik modifikatsiyalar kiritildi. Taq uning xususiyatlarini yaxshilash uchun polimeraza.

PCR kuchaytirilayotgan DNK segmentining ikkala uchidan DNK sintezining bir necha bosqichlarini o'z ichiga oladi. DNK sintezi paytida nima sodir bo'lishini eslang: bitta ipli oligonukleotidli primer DNKda bir-birini to'ldiruvchi ketma-ketlikni bog'laydi. Bu ikki tomonlama mintaqani ta'minlaydi
primerni uzaytiradigan DNK polimeraza langari, DOIM5 & ​​rsquo dan 3 & rsquo yo'nalishida sayohat qilish. Tergovchilar DNK replikatsiyasining boshlang'ich joylarini reaktsiya uchun primer sifatida xizmat qilish uchun oligonükleotidlarni etkazib berish orqali nazorat qiladilar (quyida ko'rsatilgan). Sizning sevimli geningiz Yfg). PCR primerlarini loyihalash uchun tadqiqotchilar maqsadli DNKdagi primer bog'lanish joylari uchun aniq ketma-ketlik ma'lumotlariga muhtoj. (Eslatma: Kuchaytiriladigan butun ketma-ketlik uchun ketma-ketlik haqida ma'lumot kerak emas. PCR ko'pincha ikkita ma'lum primer bog'lanish joylari o'rtasida yuzaga keladigan ketma-ketlikni aniqlash uchun ishlatiladi.) PCR uchun ikkita primer talab qilinadi. Bir primer DNK spiralining har bir ipini bog'laydi.

PCR bir necha daqiqali denaturatsiya davri bilan boshlanadi, bu davrda reaktsiya aralashmasi DNK spiralining ikkita ipini bir-biriga bog'lab turadigan vodorod aloqalarini uzish uchun etarlicha yuqori haroratda inkubatsiya qilinadi. Samarali denaturatsiya juda muhim, chunki DNK polimeraza shablon sifatida bitta torli DNKni talab qiladi. Dastlabki denatürasyon segmenti keyingi denatürasyon bosqichlariga qaraganda uzunroqdir, chunki genomik DNK kabi PCR uchun biologik shablonlar ko'pincha ko'plab vodorod aloqalari bilan birlashtirilgan uzun, murakkab molekulalardir. Keyingi PCR sikllarida oldingi sikllarning (qisqaroq) mahsulotlari ustun shablonlarga aylanadi.

Dastlabki denaturatsiyadan so'ng, PCR turli haroratlarda amalga oshiriladigan uchta segmentli 30-35 tsikldan iborat. PCR reaktsiyalari metall reaktsiya blokining haroratini tezda moslashtiradigan termosiklerlarda inkubatsiya qilinadi. Oddiy tsikl quyidagilarni o'z ichiga oladi:

  • denaturatsiya bosqichi - odatda 94 va 778C
  • primerni yumshatish bosqichi - odatda 55 ̊C
  • kengaytirish bosqichi - odatda 72 ̊C

PCR reaktsiyalariga denaturatsiya, tavlanish va cho'zilishning bir necha davrlari kiradi.

primer ketma -ketligi. DNK polimerazalari kengayish haroratida faollashadi, bu ularning optimal haroratiga yaqinroq. Tergovchilar har xil primerlar, andozalar va DNK polimerazalarini joylashtirish uchun yuqoridagi qadamlarning harorati va vaqtini moslashtiradi.

Belgilangan o'lchamdagi PCR mahsulotlari eksponent sifatida to'planadi

PCR haqiqatan ham zanjirli reaktsiya, chunki DNKning qiziqish ketma -ketligi taxminan ikki baravar ko'payadi
har bir tsikl bilan. O'n tsiklda ketma -ketlik kuchayadi

1000 marta (2 10 = 1024). Yigirma tsiklda ketma -ketlik kuchayadi

million barobar. O'ttiz tsiklda ketma -ketlikni nazariy jihatdan kuchaytirish mumkin

milliard marta. Laboratoriyada PCR reaktsiyalari odatda 30-35 tsikl denatürasyon, tavlama va cho'zishni o'z ichiga oladi. PCRni tushunish uchun birinchi bir necha tsikllarga e'tibor qaratish muhimdir. Belgilangan o'lchamdagi PCR mahsulotlari birinchi navbatda ikkinchi tsiklda paydo bo'ladi. Uchinchi tsiklda mo'ljallangan PCR mahsulotining ekspansional kuchayishi boshlanadi.

Birinchi tsiklda termostabil DNK polimerazalari DNKni sintez qilib, primerlarning 3 va uchlarini uzaytiradi. DNK polimerazalari - bu DNKdan tushguncha DNK sintezini davom ettiradigan jarayonli fermentlar. Binobarin, birinchi siklda sintez qilingan komplementar DNK molekulalari turli uzunliklarga ega. Mahsulotlarning har biri boshlang'ich pozitsiyasini aniqladi, chunki u astar ketma -ketligidan boshlanadi. Bu & ldquoanchored & rdquo ketma -ketliklari keyingi tsiklda, belgilangan uzunlikdagi PCR mahsulotlari birinchi marta paydo bo'lganda, DNK sintezi uchun shablonga aylanadi. PCR uchun boshlang'ich shablon PCRning har bir keyingi siklida nusxa ko'chirishda davom etadi va har bir tsiklda ikkita yangi &ldquoanchored&rdquo mahsulotini beradi. Chunki & ldquoanchored & rdquo mahsulotlarining uzunligi juda o'zgaruvchan, ammo ularni PCR reaktsiyasining yakuniy mahsulotlarida aniqlab bo'lmaydi.

Belgilangan uzunlikdagi DNK iplari birinchi navbatda ikkinchi tsiklda paydo bo'ladi. &ldquoanchored&rdquo bo'laklaridan replikatsiya mo'ljallangan uzunlikdagi PCR mahsulotlarini hosil qiladi. Ushbu aniqlangan uzunlikdagi bo'laklarning soni har bir yangi tsiklda ikki baravar ko'payadi va tezda reaktsiyada ustun mahsulotga aylanadi.

Ko'pgina PCR protokollari 30-35 kuchaytirish tsiklini o'z ichiga oladi. So'nggi bir necha tsikllarda istalgan PCR mahsulotlari bir necha sabablarga ko'ra endi eksponent ravishda to'planmaydi. Har qanday enzimatik reaktsiyada bo'lgani kabi, PCR substratlari tugaydi va 94 ℃ 778C da takroriy inkubatsiya davrlari denatüratsiyalana boshladi. Taq polimeraza.

Primer tavlanish PCR o'ziga xosligi uchun juda muhimdir

Yaxshi primer dizayni PCR muvaffaqiyati uchun juda muhimdir. PCR, primerlar DNK shablonidagi maqsadli ketma -ketlik uchun juda aniq bo'lsa, eng yaxshi ishlaydi. Noto'g'ri tanlash, primerlar qisman bir -birini to'ldiruvchi ketma -ketlik bilan bog'langanda sodir bo'ladi, bu DNK polimerazasini noto'g'ri DNK ketma -ketligini nusxalashiga olib keladi. Yaxshiyamki, tergovchilar, odatda, primerlarning to'g'ri nishonlar bilan gibridlanish ehtimolini maksimal darajada oshirish uchun eksperimental parametrlarni sozlashi mumkin.

PCR primerlari odatda 18 dan 25 tagacha sintetik oligonukleotidlardir. Astarni loyihalashda tadqiqotchilar uning T ni hisobga oladim, primer va shablon o'rtasida hosil bo'lgan duragaylarning yarmi eriydigan harorat. Umuman olganda, gibridning termal stabilligi primerning uzunligi va uning tarkibidagi GC tarkibining oshishi bilan ortadi. (Eslatib o'tamiz, GC-bazali juftlik AT-juftlik bilan solishtirganda uchta H-bog 'bilan barqarorlashadi.) Quyidagi formula T ning taxminiy bahosini beradi.m oligonukleotid duragaylari. Bu formulada, n nukleotidlar sonini bildiradi va bir valentli kationlarning kontsentratsiyasi molyar (M) birliklarda ifodalanadi.


Primer heterodimer muammosi - Biologiya

C2005/F2401 '10 ma'ruza #12 - DNK sintezining yakunlanishi PCR RNK va amp nima uchun yaxshi? Keyingi safar: RNK qanday yaratilgan?

Mualliflik huquqi 2010 Debora Mowshowitz va Lourens Chasin, Biologiya fanlari boʻlimi, Kolumbiya universiteti Nyu-York, NY. Oxirgi yangilangan: 19.10.2010 03:10

Tarqatma materiallar: 11-3 - DNKning replikatsiyasi - tafsilotlar Fork & amp
12-A-PCR
12-B = RNK va DNK sintezini taqqoslash
Barcha tarqatma materiallarning nusxalari har bir ma'ruzadan so'ng doktor M kabinetining tashqarisidagi qutilarga (Muddning 7 -qavati) joylashtiriladi. Barcha tarqatma materiallarning skanerlangan nusxalari joylashtiriladi, lekin ma'ruza tugashi shart emas.

Eslatmalar, muammolar kitobining joriy va oldingi nashrlari va hokazolarning barcha tuzatishlari uchun tuzatishlar sahifasiga qarang. Agar biron bir xato topsangiz, doktor M.ga elektron pochta orqali xabar yuboring.


I qism: DNK replikatsiyasi, davomi. - DNK 2-vazifani qanday bajaradi?

I. DNK replikatsiyasidagi hodisalar Fork -- Ligazaning uzluksiz sintezi va roli.

Millionlab asosiy juftlik uzunlikdagi haqiqiy DNK molekulasi bilan replikatsiya qanday ishlaydi? Oxirgi vaqtdagi muhim fikrlar (batafsil ma'lumot uchun 10-ma'ruza izohlariga qarang):

Siz butun molekulani yechmaysiz va har bir shablon ipini alohida takrorlamaysiz. Buning o'rniga, bir chekkadan boshlab, bir vaqtning o'zida ikkita spiralni bo'shating.

Barcha yangi zanjirlar shablonga parallel ravishda 5 dan 3 gacha o'sadi.

Bir yangi zanjir (etakchi ip) uzluksiz sintezlanadi va bitta yangi zanjir (ortda qolgan ip) uzluksiz sintezlanadi.

Ligaza fermenti orqada qolgan ipning bo'limlarini (Okazaki bo'laklari) birlashtiradi.

Diagrammalar uchun Sadava fig 13.16 (11.18) yoki Becker 19-9 ga qarang. Shuningdek qarang tarqatma material 11-3. Quyida sanab o'tilgan qadamlar va harflar va oxirgi marta tarqatma materialdagi yuqori diagrammaga tegishli.

Tarqatma materialdagi 5 va 6-bosqichlar quyida tushuntirilganda oxirgi marta o'tkazib yuborilgan.


II. Primerlar va Primase. (Tarqatma boshi 11-3. 5-qadam va 6-qadam)

Agar siz DNK polimeraza, ligaza, pirofosfataza, dATP, dGTP, dTTP va amp dCTP ni probirkaga (+ hamma ochilmaydigan fermentlarga) qo'ysangiz, DNK olasizmi? Yo'q, chunki DNK polimeraza yangi zanjirni boshlay olmaydi -- u faqat oldindan mavjud bo'lgan zanjirning 3' uchiga qo'shilishi mumkin. (Bir nechta DNK polimerazalari bor, lekin ularning hammasi shunday xususiyatga ega.) Xo'sh, DNKning yangi zanjirlari qanday boshlanadi? Primer va primazadan foydalanish.

B. Yechim in vivo

1. Primase Primerni qanday qiladi -- Sadava rasmiga qarang. 13.13 (11-16) yoki Bekker 19-11.

b. Astar: Primaza tomonidan yaratilgan qisqa RNK cho'zilishi primer deb ataladi. Voqealar tarqatilishida (11-3), RNK primeri nuqta bilan ko'rsatilgan. (Quyidagi diagrammada primer qizil chiziqli chiziqdir.) Primaza primer sintezini katalizlaydi, so'ngra DNK polimeraza RNK primerining 3 'uchiga qo'shiladi.

2. Primer qanday o'chiriladi va o'zgartiriladi
Primerni (qisqa RNK bo'limi) olib tashlash va uni DNK bilan almashtirish kerak. Jarayon 11-3-tarqatmalarning 5 va 6-bosqichlarida va quyidagi diagrammada ko'rsatilgan. Quyidagi qadamlarning ba'zilari bir vaqtning o'zida sodir bo'lishi mumkin, lekin jarayonni aniqroq qilish uchun alohida tasvirlangan.

5-qadam: Okazaki №2 fragmentining 3' uchi va №1 fragmentning 5' uchi o'rtasida bo'shliq qoldirib, E molekulasi hosil bo'lgan primer chiqariladi.

6 -qadam: DNK polimeraza bo'shliqni to'ldirish uchun #2 primerning 3 'uchiga qo'shilib, F molekulasini beradi.

7 -qadam: Ligaza orqada qolgan ipning bo'sh uchlariga qo'shilib, G molekulasini beradi.

RNK primerini olib tashlash (5-bosqich) va bo'shliqni DNK bilan to'ldirish (6-bosqich) bitta fermentning ikki xil katalitik qismidan foydalangan holda bir vaqtning o'zida sodir bo'lishi mumkin. DNK polimeraza mas'ul bo'lgan ferment, ammo muntazam o'sib borayotgan zanjirning 3' uchiga qo'shadigan bir xil bo'lishi shart emas. (Fermentlar bir nechta katalitik faollikka ega bo'lishi mumkin. Batafsil ma'lumot uchun pastga qarang.)

3. Foydalanishning qisqacha rasmlari va astarni almashtirish
S
ee Becker 19-13-rasm yoki Sadava-rasm. 13.17 (11.19) yoki Quyidagi rasm. Eslatma: Matnlarning eski nashrlaridagi ba'zi rasmlarda barcha tafsilotlar to'g'ri emas. Ba'zi raqamlar shuni ko'rsatadiki, DNK RNK primerini DNK polimeraza qo'shishi uchun 3 'bo'sh uchiga ehtiyoj sezmasdan o'zgartirishi mumkin. Boshqa raqamlar ligazaning Okazaki bo'laklarini noto'g'ri joyda birlashtirganini ko'rsatadi. (Quyidagi rasmga yoki 6-14-muammo yechimiga qarang, B-3-qism, bogʻlanishning toʻgʻri joylashishi. 6-14-muammodagi replikatsiya vilkasi quyidagi rasmdagi vilkadan teskari yoʻnalishda harakatlanishiga eʼtibor bering.)

Astarni sintez qilish va almashtirish jarayonini sarhisob qiladigan quyidagi rasmda barcha o'qlar 5 dan 3 gacha. Replikatsiya vilkasining faqat bir tomoni - orqada qolgan ipning sintezini bajaruvchi tomoni ko'rsatilgan. Uzluksiz sintezni amalga oshiruvchi tomon qoldirilgan. E'tibor bering, quyidagi replikatsiya vilkalari ketadi o'ngdan chapga -- DNK o'ngdan chapga ochilmoqda.

Astarni almashtirish va o'zgartirish funktsiyasi, shuningdek, 11-3-tarqatmalarga qarang.

Primerni olib tashlash va replikatsiya vilkasidagi boshqa hodisalarning animatsiyalari uchun yuqoridagi ma'ruza boshida berilgan havolalarga qarang.


C. Yakuniy muammo -- primerlarga bo'lgan ehtiyojning biologik natijasi (eukariotlarda).

1. & quotloose end & quot Muammo

Yangi ipning chap uchidagi astarni almashtirishning oson yo'li yo'q (yuqoridagi rasmda), shuningdek, Bekker fig. 19-15. RNKni olib tashlash mumkin, lekin bu bo'shliqni to'ldirish uchun hech qanday DNK qilish mumkin emas.

2. Yechimlar

a. Kichik DNKlar (va ko'pchilik prokaryotik xromosomalar) odatda dumaloq, bu muammoni chetlab o'tadi.

b. Telomerlar va telomeraza. Chiziqli xromosomalar (eukariotlardagi me'yor) har bir replikatsiya bilan qisqaradi -- keyingi aylanishda, hozirgina yaratilgan zanjir shablon bo'ladi va u asl nusxadan asl nusxadan qisqaroq bo'ladi. Chiziqli xromosomali organizmlar qanday oqibatlardan qochishadi: xromosomalardagi DNK molekulalarining uchlarida maxsus takrorlangan ketma -ketliklar (telomerlar deb ataladi) bor. Takrorlashlar telomeraza deb ataladigan ferment bilan almashtirilmasa, asta-sekin yo'qoladi. Keyingi muddatda eukaryotlarga e'tibor qaratsak, batafsilroq ma'lumotlar muhokama qilinadi.

2009 yilgi tibbiyot fanlari bo'yicha Nobel mukofoti telomer va telomerazani aniqlagan tergovchilarga berildi. Nobel mukofotining rasmiy bosh sahifasiga o'ting, kimyo va tibbiyot fanlaridagi barcha mukofotlarning tavsifiga havolalar. Ushbu mukofotlarning aksariyati ushbu kursda yoritilgan kashfiyotlar uchun berilgan.

Faqat ma'lumot: Eukaryotik xromosomalar ba'zan har bir replikatsiyada qisqaradi, lekin bu odatda muhim emas, chunki yo'qolgan bo'limlar (telomerik takrorlanishlar) genetik (kodlash) ma'lumotlarini o'z ichiga olmaydi. Sadava rasmiga qarang. 13.20 (11.21) yoki Bekker 19-16. (E'tibor bering: Purvesning 6 -nashrdagi rasmida noto'g'ri chiziq & quot; juda qisqa. & Quot; 3 'uchi teskari emas, balki 5' uchidan uzunroq bo'lishi kerak.) Telomerazaning etishmasligi oddiy somatik hujayralar bilan chegaralanishi mumkin. 50-60 bo'linishning cheklangan umr ko'rish muddati. Tuxum va sperma ishlab chiqaradigan jinsiy hujayralar telomeraza hosil qiladi, shuning uchun yangi avlod har doim to'liq uzunlikdagi telomerlardan boshlanadi. Telomeraza qanday ishlashi haqida animatsiya uchun http://faculty.plattsburgh.edu/donald.slish/Telomerase.html saytiga qarang. (E'tibor bering, bu animatsiya eukaryotlar uchun mo'ljallangan, bu holda etakchi va orqada qolgan iplar uchun ikki xil DNK polimeraza mavjud. Ikkalasi ham zanjirlarni 5 dan 3 gacha yo'nalishda o'sadi.)

Astarlarni ko'rib chiqish uchun 6-12-muammo, A-D va 6-14-muammolarga qarang. Agar sizda 2004 yilgi nashr bo'lsa. muammoli kitobning 6-12-muammolarining D qismida xatolik bor. Keyingi nashrlar tuzatilgan.

D. DNK polimerazasining katalitik faolligi

1. DNK polimerazalari murakkab fermentlardir. DNK polimerazalari bir nechta bo'linmalarga (peptid zanjirlari) va bir nechta fermentativ faollikka ega. Turli xil fermentativ faolliklar bir xil fermentning turli bo'linmalari yoki turli fermentlar tomonidan katalizlanishi mumkin. Bu sinfda biz barcha DNK polimerazalarini birlashtiramiz va ularni bitta ferment sifatida ko'rib chiqamiz. Murakkab sinflarda turli DNK polimerazalarining xossalari ajratiladi.

2. Katalitik faollik nechta?

DNK polimerazalari kamida ikki xil katalitik faollikka ega:
(1) polimeraza: dXTP yordamida o'sayotgan zanjirning 3' uchiga qo'shiladi va PPni chiqaradii.
(2) 5' dan 3' eksonukleaza: gidroliz orqali primerning 5' uchidan nukleotidlarni olib tashlaydi.

DNK polimerazalari qo'shimcha katalitik faollikka ega bo'lishi mumkin:
(3) 3' dan 5' eksonukleaza: gidroliz orqali o'sayotgan zanjirning 3' uchidan nukleotidlarni olib tashlaydi. Bu fermentga korreksiya qilish imkonini beradi -- yangi hosil qilgan fosfodiester bog'lanishlarini gidrolizlash orqali xatolik bilan qo'shilgan nukleotidlarni "zaxiralash" va olib tashlash (agar noto'g'ri asos o'rnatilgan bo'lsa). Zaxiralanganda, DNK pol. quyidagi reaktsiyani katalizlaydi:

rxn A: zanjir (uzunligi n+1 birlik) + H2O ↔ zanjiri (n uzunlikdagi birlik) + XMP

3 dan 5 gacha ekzonuklez DNK sintezi paytida xatolarni tuzatish va yuqori aniqlikni saqlashda muhim ahamiyatga ega. E'tibor bering, A reaktsiyasi polimeraza reaktsiyasining teskarisi emas. Quyidagi 4 ga qarang.

3. Terminologiya: Nukleotidlarni zanjir oxiridan birma -bir olib tashlash qobiliyati ekzonukleaza faolligi deyiladi. (exo = tashqi yoki oxiridan). Eksonukleazning ikki turi mavjud:

a. 3' dan 5' ekzo. DNK -polimerazaning fermentativ qobiliyati dalillarni o'qishda ishlatiladi, nukleotidlarni zanjirning 3 'uchidan birma -bir olib tashlaydi. Shuning uchun u 3' dan 5' gacha ekzonukleaza faolligi deb ataladi.

b. 5 'dan 3' gacha exo. RNK primerini olib tashlaydigan DNK polimerazasining fermentativ faolligi boshqa eksonukleaza faolligiga ega - bu ferment nukleotidlarni primerning 5 'uchidan (3' uchidan emas) birma -bir olib tashlaydi. 5 dan 3 gacha ekzonukleaza faolligiga ega.

4. 3' dan 5' gacha bo'lgan ekzonukleaza reaktsiyasi polimerlanish reaktsiyasining teskarisi bilan bir xil emas.

Bu erda DNK polimeraza tomonidan katalizlangan normal cho'zilish reaktsiyasi (o'ngda):

rxn B: zanjir (n uzunlik birlik) + XTP ↔ zanjir (n+1 uzunlik) +PPi

Substratlar va mahsulotlarning to'g'ri konsentratsiyasini hisobga olgan holda, har qanday ferment o'z reaksiyasini har ikki yo'nalishda ham katalizlay oladi. Polimeraza reaktsiyasini qaytarish pirofosfatni qayta qo'shish va dXTPni qayta tiklash orqali fosfodiester bog'lanishini buzishni anglatadi:

(rxn B chapda): Zanjir (n +1 birlik uzunlikda) +PPi ↔ Zanjir (n uzunlikdagi birlik) + XTP

Biroq, 3' dan 5 gacha bo'lgan ekzo katalizlaydigan narsa rxn B ning teskarisi emas (rxn B chapga) - bu fosfodiester bog'lanishining gidrolizi (rxn A). Yangi hosil bo'lgan fosfodiester aloqasi bo'ylab gidrolizlash yoki suv qo'shganda dXMP (dXTP emas) chiqariladi. 3 'dan 5' gacha eko, agar u noto'g'ri bo'lsa, lekin dXTPni qayta tiklamasa, oxirgi nukleotidni olib tashlaydi. Shuning uchun gidroliz polimeraza reaktsiyasini qaytarishdan farq qiladi.


III. Ikki tomonlama replikatsiya. (Tarqatma materialining pastki qismi 11-3). Ko'p replikatsiya vilkalar

A. Bir DNKda nechta replikatsiya vilkalari? Vilkalar qancha ko'p bo'lsa, replikatsiya shunchalik tez bo'ladi. Ko'pgina kichik genomlar (masalan, bakterial va virusli DNK) dumaloq bo'lib, ikki tomonlama takrorlanadi-2 vilkalar bitta manbadan chiqadi, 11-3 tarqatma varaqning pastki qismida yoki Sadava fig. 13.19A (11.13A) yoki Bekker 19-4 (19-5). Uzunroq DNK molekulalari odatda chiziqli bo'lib, Sadava rasmida ko'rsatilganidek, ko'pincha ikki tomonlama ko'payish kelib chiqishiga ega. 13.19B (11.14B) yoki Bekker rasm. 19-5 (19-6) -- bu haqda keyinroq eukaryotlarga kelganimizda muhokama qilinadi.

B. Ikki tomonlama Replikatsiya qanday ketadi? Tarqatma varaqdagi yuqori rasmda sizda DNK bo'ylab harakatlanadigan bitta vilka yoki fermuar bor. Pastki rasmda sizda 2 ta fermuar yoki vilkalar bor. Ikkalasi ham bir nuqtadan boshlanadi (nuqta chiziq = DNK replikatsiyasining kelib chiqishi = ori), lekin bitta vilka chapga, bitta vilka o'ngga ketadi. Har bir vilkadagi voqealar tarqatma materialning yuqori qismida ko'rsatilganlar bilan bir xil, ammo vilkalar pastga emas, chapga va o'ngga boradi. Har bir vilkada siz avvalgidek, bir ipda bo'shashmasdan, uzluksiz sintez, ikkinchisida esa uzluksiz sintez va ampliatsiyaga ega bo'lasiz. If the DNA is circular, the right fork is really going clockwise and the left fork counterclockwise, and the 2 forks proceed until they meet in the middle of the molecule, approximately 180 degrees from where they started. (See Becker fig. 19-4 (19-5).)

C. An Important Definition: Bidirectional replication means that there are 2 forks that move in opposite directions. It does NOT refer to the fact that the 2 DNA iplar (leading and lagging strands) are made in opposite directions. That is called uzluksiz synthesis, and it always happens at every fork whether there is one fork (unidirectional replication as in the top panel of handout 11-3) or two (bidirectional replication as on the bottom of the handout.)

To be sure you understand what is happening in the bottom picture, it is a good idea to write in all the 5' and 3' ends on the DNA's shown and also to number the Okazaki fragments at each fork to show the order in which they are made.

To review bi-directional replication, see problem 6-13, part A.


Part II -- PCR (Handout 12A) -- Note: Handout 12A was revised on 10/18, and the steps in B below were re-written to match the revised version. You should reprint B below if you printed it before 10/18/10. The remainder is unchanged.

IV. PCR (Polymerase Chain Reaction) A Practical Application of the need for Primers.

The inventor, Kary Mullis, received the Nobel prize in 1993. For his acceptance speech, biography, etc. see the Nobel Prize official site. For uses of the technique, see class handout.

A. Idea of prefab primer, hybridization.

DNA synthesis will not start without a primer. In a living cell, primase (a type of RNA polymerase) makes the necessary RNK astar. Then DNA polymerase can take over, adding on to the 3' end of the primer. In a test tube, you can omit primase and use an oligonucleotide (short polynucleotide, usually DNA) as primer (= prefab DNA primer) to force replication to begin wherever you want. The primer you add will hybridize to its complementary sequence, wherever that happens to be (not necessarily at the end of the DNA) and DNA polymerase will add on to the 3' end of the primer, thereby starting elongation of a chain from wherever the primer is.

B. Steps of PCR -- see PCR handout (12A), Sadava fig. 13.22 (11.23), and/or Becker Box 19 A. For an animation, go to http://www.dnalc.org/ddnalc/resources/pcr.html

The site listed above (The Dolan DNA Learning Center) has many good features you may want to check out. There is a list of additional animations on PCR, DNA replication, etc. at http://www.dna.utah.edu/PCR_Animation_Links.htm. Please let Dr. M know if you find any of these sites (or any others) particularly useful.

1. First Cycle: You take your template (A) and denature it. (Step 1 = denaturation results in B.) Then you add primers (one to each strand) to the denatured DNA and cool the mixture. When you cool the mix down, each oligonucleotide primer hybridizes to its complement. (Step 2 = hybridization to primer results in D*.) Under the conditions used, the two long strands of template do not renature to each other. Then the DNA polymerase adds on to the 3' end of primer until it reaches the end of the template strand. (Step 3 = elongation results in E.) This completes the first cycle (ends at E). The new strands you just made (dashed on handout in E) include the target sequence, plus some extra DNA on their 3' ends. (This "extra" corresponds to the sequence between the target area and the 5' end of the shablon strand.)

*Note: There is no (C) on the handout to avoid confusion with Watson (W) and Crick (C) strands.

2. Second Cycle: Same procedure as before in cycle 1. You heat the DNA to denature it (step 4 = step 1), and add more of the same primers as before (step 5 = step 2). Then you allow DNA polymerase to add on to the 3' ends of the primers (step 6 = step 3). This completes the second cycle (ends at H). On the handout, only the fate of the new strands made in cycle two is shown after F. The old strands simultaneously go through another cycle just like the one above (steps 2 & 3), but this is not shown on the handout. The new strands you made in cycle 2 (shorter strand of each molecule of H) include only the target sequence.

3. Third Cycle: Same procedure as before in cycles 1 & 2. You heat the DNA again to denature it (step 7), add primers (step 8) and allow DNA polymerase to add to the primers (step 9. This completes the 3rd cycle (ends at K). On the handout, only the fate of the new strands made in cycle two is shown after I. (The fate of the complementary strands, left over from the previous cycle, is to repeat steps 5 & 6.) At the end of this cycle, you finally have double-stranded DNA molecules the length of the target sequence (see K).

4. Additional Cycles: Same procedure as in previous cycles (repeat of steps 1-3). After each cycle you heat the reaction mixture to denature the DNA, and then you cool the mixture down to start the next cycle. In each cycle, primer sticks to the appropriate spot (its complement) and polymerase starts at the 3' end of the primer and goes to the end of the template. Note that primers are complementary to sequences in the middle of the original chain, but that after two cycles the parts beyond the primers are no longer copied. .

5. How reaction is actually carried out . All components (template and excess of heat resistant polymerase, primers & dXTP's ) are present from the very beginning. The mixture is heated and cooled repeatedly to end and start subsequent cycles. You don't have to add primers, polymerase, etc. to start each cycle.

6. How many Primers? New molecules of primer are used in each round. However, the primer molecules used in each round have the same sequences as the ones used in all the previous rounds. The primers are not reused -- new primers (with the same sequences as before) are needed for each cycle. You need only two types (sequences) of primer, but you need many molecules of each, just as you need many molecules of dATP, dTTP, etc.

7. Identification of Product. The products of the PCR reaction are usually identified by their lengths, which are determined by gel electrophoresis without SDS. (Why no SDS needed? Think about it.) Gels are used that separate DNA molecules on the basis of their molecular weights (which depends on chain length). Hybridization to labeled probes is often used to detect the positions of the bands of DNA on the gel. (More on this later.) An animation of DNA gel electrophoresis is at http://www.dnalc.org/ddnalc/resources/electrophoresis.html.

C. Special Features of PCR (as vs. regular DNA synthesis)

1. Special Polymerase. The DNA polymerase used in this procedure is a special heat-resistant one (called Taq polymerase) that is not denatured when the temperature is raised to separate the two strands of the DNA. This special polymerase was isolated from bacteria that live in a hot spring.

2. No replication fork or discontinuous synthesis. Note that the entire template molecule is denatured (or 'unzipped') completely before each cycle, so the complement to each strand can be made continuously. There is no replication fork and thus no discontinuous synthesis here.

3. Preformed DNA primer. Primase is absent, so no RNA primers are made. Oligonucleotides of DNA (not RNA) are added instead to act as primers.

To review the PCR technique, see problem 6-13, C-1 and 6-15.

For an animation of PCR and links to animations of other DNA techniques, see the urls listed above or go to the links page.)

1. Amplification: Uses small number of starting molecules & produces large number of copies of target sequence. You need amplification to get enough target DNA to hybridize to a probe.

The beauty of this scheme (PCR) is that the desired (target) sequence is copied exponentially and the other parts of the original DNA are copied linearly. So after a few cycles you have lots of copies of the target sequence (and not much of anything else). To convince yourself of this, see the answer to problem 6-13, part C-2. To use this technique and make many copies of the target sequence all you need (in theory) is ONE starting DNA molecule (and appropriate primers). Given current technology, you need 10-50 starting DNA molecules. You can use the multiple copies for many different purposes such as characterization and/or identification as explained below. Before PCR, you couldn't get enough DNA to do chemical tests, so you couldn't compare different DNA samples.

2. Detection -- Can be Used to see if a particular target DNA is present or not.

You can add primers to a sample that you suspect contains some particular target DNA, such as HIV DNA, or DNA from genetically modified corn, or DNA from pond water. The primers are complementary to a sequence found only in the target DNA -- the one you are testing for. (In the cases mentioned, the primers would be complementary to a sequence in HIV DNA, or a sequence added to ordinary corn DNA by genetic engineering methods to make the special corn, or to a DNA sequence unique to American bullfrogs.) Then you see if polymerase can make DNA. If no target DNA is present, primers will have nothing to hybridize to, so polymerase will have nothing to add on to, and no copies of DNA will be made. So if you qilma get multiple copies, it indicates there was nothing to copy -- your target DNA was not there. Agar Siz qilmoq get multiple copies, your target DNA was in the sample.

Notes: (1) The standard HIV screening test is not for HIV itself or for HIV DNA but for antibodies to proteins of HIV. (PCR is used as a backup to confirm a positive result with the standard screening test, or to measure the actual levels of HIV.)

(2). Why would you test for genetically modified corn? StarLink corn is a type of genetically modified corn that was approved for animal feed, but not for human use. In spite of attempts to keep it separate, it has turned up in many human foods. It is probably harmless to humans, but no one wants to take any chances. Testing for the modified DNA is the only way to tell if StarLink corn (or any other genetically modified food) is present in a mixture or not. A site with an explanation of the StarLink fiasco is at http://www.geo-pie.cornell.edu/issues/starlink.html.

3. Forensics -- Can be Used for identification -- DNA fingerprinting

a. Asosiy fikr: PCR can be used to copy specific sections of the DNA from different samples -- for example, from DNA left at the scene of a crime and from DNA from a suspect. The sections of amplified DNA can then be compared to see if they match or not (in length, sequence, etc.). The sections that are compared are highly variable ones that probably don't carry any information and are merely spacers in the DNA. If enough sections are checked, you can determine (to a very high degree of certainty) whether the two DNA samples came from the same person or not. DNA testing can be used to identify the guilty (inclusions) and to clear the innocent (exclusions). Alec Jeffreys, who first came up with the idea of using DNA testing for identifications, received a Lasker award in '05. For a pdf with details see the Lasker site.

b. Misollar: See articles handed out in class and article from the San Francisco Chronicle of 10/19/99. (Note: you'll need to go to the SFChronicle web site itself if you want to see the pictures or get some of the older articles.)

c. Inclusions: If the samples match at enough highly variable spots, then there is a very high probability the samples came from the same person, because the degree of variation is so high that only a few different people in the world should have the same pattern.

d. Exclusions: If the two samples do not match, then it is clear that the two samples came from different individuals and the suspect could not have committed the crime (since the DNA at the scene came from someone else).

e. STR's: The variable sections that are tested are often ones that have different numbers of short tandem repeats (STR's). The primers hybridize to regions outside the section with the repeats. The number of repeats in each DNA can be figured out from the length of the sections amplified by PCR. The new FBI data base contains the information from checking 13 sections with variable numbers of STR's.

For a great site from the Dolan Learning Center with examples of how DNA is used for identification and forensics click here.

4. Bar Coding (See the article on handout B from lecture 10. For more details on Fish bar coding, see the FishBol site.)

The tests of the DNA from different organism used a similar principle to the one used in forensics. A particular gene that varies from species to species was amplified and then sequenced. The procedure is called 'Bar coding' because the sequencing procedure produces a pattern that looks like a supermarket bar code. There is enough variation in the sequence (or Bar code) of that particular gene to identify the species of animal or fish from which it came. In this case, the the amplified DNA from the different samples is compared to the DNA from reference samples. The actual base sequences of the various DNAs are compared. In forensics, the amplified DNA from the crime scene is compared to the amplified DNA from the suspect, and the comparisons are based on the lengths of the amplified fragments (not on their actual sequences).

5. Why you can't do this with proteins

There are very sensitive tests for presence of proteins (usually using the catalytic activities of enzymes and/or binding abilities of antibodies), but no way to amplify (make copies of) what you detect. You can't make more protein from a protein template. PCR takes advantage of fact that DNA replicates for a living to make more copies. Siz mumkin make more DNA from a DNA template.

Note: So-called DNA fingerprints are characteristic of the person/DNA from which they came. So-called protein fingerprints are characteristic of the oqsil from which they came. That's why both are called 'fingerprints.' However the two types of 'fingerprints' are made differently and used for different purposes.


Part III -- How does DNA do job #1? How does 'DNA make Protein?'

V . Central Dogma -- How does DNA do job # 1?

A. Big Picture . So we have a big DNA that includes a particular gene = stretch of DNA coding for a single peptide how will we make the corresponding peptide?

Note: gene usually means a stretch of DNA encoding 1 polypeptide, but there are complications as we'll see later.

1. Basic idea -- see also Becker fig. 21-1 or Sadava fig. 14.2 (12.2 & 12.3):

  • Replication = DNA synthesis using a DNA template.

  • Transcription = RNA synthesis using a DNA template.

  • Translation has two possible meanings (we will stick to the first):

    (1) Usual meaning = protein synthesis using an RNA template (RNA → protein). Used in contrast to transcription (DNA → RNA).
    (2) In some contexts, translation can mean the entire process (DNA → RNA → protein).

1. Structure: Sadava rasmiga qarang. 4.2 (3.24) and table 4.1 (3.3) yoki Becker table 3-5 & fig. 3-17 for comparison of DNA and RNA. RNA is single stranded (although sections may double back on themselves → double stranded regions), has U not T, ribose not deoxy and is generally shorter, but otherwise like DNA. RNA is less stable than DNA -- more easily damaged (because of reactive OH on ribose and because a single strand is more exposed) va less easily repaired (because no 2nd strand to use to correct mistakes on first strand). DNA is also more easily repaired because it has T not U, so damaged C's (which are oxidized to U) can be recognized and removed. Qisqa bayoni; yakunida:

DNA RNK Significance/Effect of Difference

Double Stranded

Single Stranded*

For RNA: Ease of repair down likelihood of damage up.

T not U

U emas T

For DNA: Ease of repair of damaged (oxidized) C up. (Damage that coverts C to U can be detected & repaired.)

Deoxyribose

Riboza

For RNA: Reactivity up, stability down

Very long

Relatively Short


For RNA: Less Information carried per molecule but molecule is much more convenient size

* RNA is basically single stranded, but can fold back on itself to form hairpins -- short regions that are double stranded. Sadava rasmiga qarang. 4.3 (3.25)

2. Synthesis. RNA grows just like DNA by adding nucleoside triphosphates (XTP's) to the 3' end of a growing chain. For RNA, enzyme for elongation is called RNA polymerase, XTP's are ribo (not deoxy) and U replaces T. Details to follow.

3. Types. There are 3 major types of RNA involved in translation: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). The roles of the different types of RNA are outlined below and will be explained in detail next time.


VI. Why mRNA?

A. Basic idea : mRNA = Working, disposable copy vs DNA = archival, permanent master copy. DNA = big fat comprehensive reference book or complex web site. mRNA = Xerox of one (book) page or print out of one web page with information you need for a particular assignment. Book stays safe in library web site remains unchanged. Xerox goes to your room, is actually used, gets covered with coffee stains, smudged, and thrown away.

B. How function of mRNA corresponds to structure

1. Convenience. Small size (1 or a few peptides' worth) is much more convenient than many genes' worth. Xerox of one page much more convenient to work with than big fat book.

2 . Preserve Master. Using mRNA to make protein saves wear and tear on master -- no coffee stains on the archival copy (DNA).

3. Flexibility. D ifferent amounts of mRNA can be made when you need to make different amounts of different proteins. More on this when we get to regulation (operons).

C. Summary: How does RNA make protein?

1. "RNA makes protein" means two things:

  • mRNA to act as template -- determines order of amino acids

  • tRNA to carry the amino acids to the template, and line them up

  • rRNA (in ribosomes) to align the tRNA's carrying the amino acids and hook the amino acids together

  • Of course you need additional proteins (enzymes and other factors) to make protein

2. Hardware vs. Software . rRNA and tRNA are the hardware or tools or machines mRNA is the software or working instructions or tapes/CDs/punchcards. Cells use same old hardware and constantly changing, up to the minute, supply of new software.


VI. Where does RNA come from?
You need lots of RNA to make protein -- tRNA, rRNA & mRNA. How do you make the RNA? All RNA is transcribed from a DNA template. Sadava rasmiga qarang. 14.4 (12.5) or Becker fig. 21-8 (21-9) & 21-10 (21-11).We'll go over how the RNA is made , and then consider how the RNA is used to make protein.

The live lecture in '09 (#12) ended here. If we don't get to it in #12, Topic VII will be covered in lecture #13.

VII. DNA synthesis vs RNA synthesis. The easiest way to go over RNA synthesis, given that we've discussed DNA synthesis at length, is to compare DNA and RNA synthesis. See handout 12-B.

A. Basic mechanism of elongation is the same:

1. Use nucleoside triphosphates (ones with ribose not deoxyribose, but mechanism same) & split off PPi use pyrophosphatase.

2. Chain grows 5' to 3' by addition to 3' end .

3. Need anti-parallel DNA template , put in complementary bases -- A (in template) pairs with U not T, but otherwise same

4. All RNA molecules (mRNA, tRNA and rRNA), not just mRNA's, are made from a DNA template. tRNA and rRNA molecules are emas made from an "mRNA" template.

See problems 7-1 & 7-2.

  • Growth of DNA chain is catalyzed by DNA polymerase (and associated enzymes)

  • Growth of RNA chain is catalyzed by RNA polymerase.

  • RNA pol. uses ribonucleoside triphosphates.

  • DNA pol uses deoxyribonucleoside triphosphates.

  • DNA is long and double stranded

  • RNA is short and single stranded

  • Template = short section, one strand at a time (for RNA synth.) vs all of both strands (for DNA synth.)

  • Nega? Because starts and stops are different. Starts & stops = sequences in DNA recognized by the enzymes = places where replication or transcription starts (or ends). These must be different for the two enzymes.

  • Names of start sequences = section where polymerase binds
    Starts for DNA synthesis = Origins. DNA pol. recognizes (binds to) start signals for replication called origins (ori's).
    Starts for RNA synthesis = Promotors. RNA pol. recognizes (binds to) start signals for transcription called promotors (P's).

See problem 7- 6

  • DNA synthesis: Replication fork moves down DNA making complements to ikkalasi ham strands one new strand is made continuously and one discontinuously. Ligase is needed for synthesis of lagging strand.

  • RNA synthesis: RNA polymerase moves down DNA making complement to one strand yoki the other (in any particular region). Therefore RNA synthesis is continuous and doesn't need ligase.

See problems 7-3, 7-4, 7-8 & 7-9.

Next time: We'll finish RNA synthesis vs DNA synthesis, and then consider how the RNA that was transcribed is translated -- how it's used to make protein.

Copyright 2010 Deborah Mowshowitz and Lawrence Chasin Department of Biological Sciences Columbia University New York, NY


Global Sensitivity Analysis. The Primer

Mathematical models are good at mapping assumptions into inferences. A modeller makes assumptions about laws pertaining to the system, about its status and a plethora of other, often arcane, system variables and internal model settings. To what extent can we rely on the model-based inference when most of these assumptions are fraught with uncertainties? Global Sensitivity Analysis offers an accessible treatment of such problems via quantitative sensitivity analysis, beginning with the first principles and guiding the reader through the full range of recommended practices with a rich set of solved exercises. The text explains the motivation for sensitivity analysis, reviews the required statistical concepts, and provides a guide to potential applications.

  • Provides a self-contained treatment of the subject, allowing readers to learn and practice global sensitivity analysis without further materials.
  • Presents ways to frame the analysis, interpret its results, and avoid potential pitfalls.
  • Features numerous exercises and solved problems to help illustrate the applications.
  • Is authored by leading sensitivity analysis practitioners, combining a range of disciplinary backgrounds.

Postgraduate students and practitioners in a wide range of subjects, including statistics, mathematics, engineering, physics, chemistry, environmental sciences, biology, toxicology, actuarial sciences, and econometrics will find much of use here. This book will prove equally valuable to engineers working on risk analysis and to financial analysts concerned with pricing and hedging.


Videoni tomosha qiling: BESHARIQNING BESH MUAMMOSI (Avgust 2022).